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Acatalytic Carbonic Anhydrases (CAs VIII, X, XI)
CHAPTER 13
Acatalytic Carbonic Anhydrases (CAs VIII, X, XI) Claudiu T. Supuran*,**, Clemente Capasso*** *Università
degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Sesto Fiorentino, Florence, Italy Department, Sezione di Scienze Farmaceutiche, Sesto Fiorentino, Florence, Italy di Bioscienze e Biorisorse, CNR, Napoli, Italy
**NEUROFARBA ***Istituto
Contents 13.1 Introduction 239 13.2 Primary sequence analysis 240 13.3 Three-dimensional structure analysis 240 13.4 Physiological function and tissue distribution of CAs VIII, X, and XI 242 13.5 CARPs’ biochemical properties after restoring the catalytic site 242 13.6 Phylogenetic analysis 243 13.7 Conclusions 244 References 244
13.1 INTRODUCTION Most structural and regulatory proteins in eukaryotes are members of a gene family. Over the course of evolution, some duplicate genes were short-lived, losing functionality, and ultimately were removed (1–5). Enzymes having a chemical requirement for invariant amino acids in the active site are particularly vulnerable to selection pressure. The positive selection more often acts on residues adjacent to, rather than directly at, a critical active site of an enzyme, and on flexible regions rather than on rigid structural elements of the protein (1,2,5). This pattern might be a general mechanism for functional diversification of enzyme families, as it allows the acquisition of new functions without disrupting the native folding structure and primary enzyme function. But the natural evolution of enzymes can eventually lead to the formation of a new active site on the protein framework, or by transformation of an old active site for a new function. Interestingly, using sequence similarity programs, it is possible to identify proteins evolutionarily related to enzymes but lacking catalytic activity due to disruption of their active site. Most of these proteins develop a new function, for example, as transcription regulators (2,4,5). In this case, the direction of the evolution is simply that the enzyme lost its activity but acquired a new function.
Carbonic Anhydrases as Biocatalysts. DOI: 10.1016/B978-0-444-63258-6.00013-5 Copyright © 2015 Elsevier B.V. All rights reserved
239
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Carbonic anhydrases (CAs) are found in almost all organisms and are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3− (6–9). In mammals, a-CA gene family consists of 13 isoforms that are catalytically active and 3 isoforms that are catalytically inactive lacking the CO2-hydratase activity as well as other known catalytic functions normally associated with these enzymes, such as the esterase activity (7,10–14). Due to the high sequence and structural similarity with the active a-CAs, these proteins have been designated as CA-related proteins (CARPs) and were originally identified by screening Purkinje cell–specific genes (7,15–18).The three mammalian CARPs are named as CARP VIII, CARP X, and CARP XI. CARPs are predicted to be composed of 291 amino acids, and have an acidic amino acid cluster of 16 Glu and 4 Asp residues within the N-terminal 50 amino acid residues (18–20).These a-isoforms have a central CA motif, but the catalytic inactivity is due to lack of one or more of the three histidine residues that are necessary for coordination of the zinc ion within the active site (18,21).The enzymatic activity of mammalian CARP isoforms can be regained by restoration of these histidine residues (18). In the family of protein tyrosine phosphatases (PTPs), there are two receptor-type PTPs, -PTPR and g-PTPR, also containing an N-terminal CA-like domain, called “CARP XVI domain” (15,17). The physiological roles of CARPs are poorly understood for the moment. Here, we account the recent data on CARPs VIII, X, and XI.
13.2 PRIMARY SEQUENCE ANALYSIS Although the CARPs show a high degree of homology with the other known a-CA isoforms, they distinguish themselves from the catalytically active CAs by a salient feature: they lack one, two, or all three zinc protein ligands from the enzyme active site, that is, His94, His96, and His119 (the hCA I numbering system is used throughout this work) (15,17–19). Figure 13.1 shows the sequence alignment of hCA II with CARP VIII, CARP X, and CARP XI (denominated from now on as hCAs VIII, IX, and XI, respectively). Residue 94 is not a His, but an Arg in all the acatalytic isoforms; residue 96 is His in hCA VIII and hCA X, but Leu in hCA XI, whereas residue 119 is His only in hCA VIII, being a glutamine in hCA X and XI.Thus, in the acatalytic isoforms, one (hCA VIII), two (hCA X), and three (hCA XI) of the protein zinc ligands are changed from His (conserved in all the other catalytically active isoforms) to another amino acid, which normally has no possibility to coordinate the catalytically crucial zinc ion within the active site (18).
13.3 THREE-DIMENSIONAL STRUCTURE ANALYSIS In 2009, Oppermann and coworkers (22) solved the X-ray crystal structure of hCA VIII at high resolution, showing that the three-dimensional fold of the protein is rather similar to that of hCA II, the most studied and physiologically dominant
Acatalytic Carbonic Anhydrases (CAs VIII, X, XI)
Figure 13.1 Sequence alignment of hCA II (accession number: AAH11949.1), hCA VIII (accession number: AAI08930.1), hCA X (accession number: AAH47456.1) and hCA XI (accession number: AAH02662.1). The catalytic residues His94, His96 and His119 in hCA II and the corresponding residues of the acatalytic CARPs are underlined. The proton shuttle amino acid (His64) is conserved in all these molecules, while the gate keeper residues (Glu106 and Thr199) are conserved only in hCA II, hCA VIII and hCA X. Legend: (*) indicates identity at all aligned positions; (:) relates to conserved substitutions; (.) means that semi-conserved substitutions are observed. The hCA I numbering system was used. Multialignment was performed with the program MUSCLE, version 3.7
catalytically active isoform. As indicated in Figure 13.2A, a chloride ion has been found in the position where the Zn(II) ion is present in the catalytic CAs, such as hCA II (Figure 13.2B).This anion participates in two hydrogen bonds with His96 and His119, and another one with a water molecule, situated in a position, which is very similar to that of the zinc-coordinated water molecule in hCA II. Arg94 from hCA VIII had no interactions either with the chloride anion or with the above-mentioned water molecule, but the shape of the “active site” cavity was very similar to that of hCA II (18,22).
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Figure 13.2 (A) Ribbon diagram of hCA VIII structure. Relevant residues (His96, His119, and Arg94), Cl− ion (gray sphere), and the hydrogen-bonded water molecule are also shown. (B) Ribbon diagram of hCA II structure (PDB code 1CA2). The three histidines, the zinc ion (gray sphere), and the zinc bound water molecule are shown.
13.4 PHYSIOLOGICAL FUNCTION AND TISSUE DISTRIBUTION OF CAs VIII, X, AND XI The physiological role of some of these proteins, which are highly conserved in mammals, seems to be quite important (10,12–14,18). Thus, CA VIII has been initially identified in the brain, mainly in the Purkinje cells, but its expression has also been demonstrated in several other organs, such as the liver, lung, heart, gut, thymus, and kidneys (16,20,23–26). CAs X and XI are also predominantly found in the brain. The strong expression of CA VIII in the cerebellum suggested a role in brain function (10,19,20). This has been indeed documented later (27,28), when a mutation in the CA VIII gene was shown to be associated with ataxia, mild mental retardation, and quadrupedal gait in humans, and with lifelong gait disorder in mice, proving the important, yet poorly understood physiological role of this protein. Furthermore, CA VIII has been reported to be overexpressed in certain tumors, including non-small lung cancer and colorectal cancer (10,19,20). CA VIII mRNA was significantly expressed in developing human lungs but to a much lesser extent in normal adult lungs (29).These findings suggest that CA VIII plays a role in cell proliferation and carcinogenesis, in at least lung and colorectal epithelial cells. CA XI has also been observed to be overexpressed in gastrointestinal stromal tumors, promoting their proliferation (30). These data strongly suggest that CARPs may possess important physiological roles, poorly understood at this moment.
13.5 CARPs’ BIOCHEMICAL PROPERTIES AFTER RESTORING THE CATALYTIC SITE Recombinant proteins of human and mouse CA VIII have been reported to lack not only CO2 hydration activity but also esterase activity toward 4-nitrophenyl acetate as substrate (18). It is of interest that the single mutagenesis at position 94 from arginine to
Acatalytic Carbonic Anhydrases (CAs VIII, X, XI)
histidine was shown to restore the CO2 hydration activity. Further mutations, leading to a more “CA-like” active site cavity (e.g., Arg94His, Glu92Gln, Ile121Val, Ile143Val), resulted in a further increase of the catalytic activity of the mutant CA VIII (18). Sitedirected mutagenesis experiments in all three CARPs, as reported earlier for CA VIII by Lindskog and coworkers and Bergenhem et al. (21,31,32), were then performed, restoring their zinc-binding His residues. Interestingly, the recombinant hCAs VIII, X, and XI possessed a high catalytic activity for the physiological reaction, CO2 hydration to bicarbonate and protons, with the following kinetic parameters: kcat ranging from 7.20 × 105 to 1.15 × 106 s−1, KM in the range of 7.1–8.3 mM, and kcat/KM in the range of 1.01 × 108 to 1.38 × 108 M−1 s−1 (18). hCA X was found to be the most active among the investigated new enzymes. It was 92.0% as effective as hCA II as a catalyst for the physiological reaction. hCA XI showed a catalytic activity intermediate between that of hCA VIII (the least active) and hCA X (the most active isoform), being 82.6% as active as hCA II.The excellent catalytic activity of the mutated CAs VIII, X, and XI, much higher than that of hCA I, may be explained considering that in these enzymes (after the change of the residues in positions 94, 96, and 119 to His residues), the proton shuttle residue (His64) is also present. Restored CARPs were inhibited in the millimicromolar range by inorganic anions, sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid (18). Among the three new isoforms, hCA X showed the highest affinity for anion inhibitors (KI of 3.6–68 mM for phenylboronic acid, sulfamic acid, sulfamide, cyanide, and azide). hCA VIII was poorly inhibited by halides, cyanate, nitrate, and sulfate (KI of 38.4–65.4 mM), whereas CA XI had a behavior intermediate between that of hCA VIII and X, regarding the sensitivity to anion inhibitors (18). Probably the anions investigated in that study bind to these enzymes in a similar manner as they bind to other a-CAs investigated in more detail, coordinating to the metal ion, with the Zn(II) ion in either tetrahedral or trigonal bipyramidal geometries (33,34).
13.6 PHYLOGENETIC ANALYSIS CARP-like sequences have been identified in mammals and invertebrates. Vaccinia virus transmembrane protein D8 was reported to contain a CA-like N-terminal domain. CARP VIII sequences have been discovered in mammals and in many deuterostome invertebrates, such as mollusks and chordates, but not in protostomes, such as insects (16,20). CARP X–like proteins are present in vertebrate and invertebrate. In the invertebrate group, CARP X–like proteins are present even in nematodes and insects. CARP XI, the last to be identified, was recognized only in tetrapod vertebrates (15,17). Recently, Parkkila and coworkers reported that there is no ortholog of CA11 in the known fish genomes, but instead there are two descendants of CA10, namely, CA10A and CA10B12. This group carried out a phylogenetic analysis on all CARPs. They found that the origin of CA8 is more ancient than the origins of CA4, CA5, CA6, CA9, CA12, and CA14, which were formed during the radiation of jawed vertebrates, or CA1, CA2, CA3, and CA13, which are specific to the tetrapod lineage (15,17,19).
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Phylogenetic analysis of CARP X sequences showed that they form two groups: one consisted of sequences from mammals, frogs, and lizards; and the other, formed by two smaller groups, consisted of sequences from fishes (17). CARP XI phylogenetic analysis suggests that the CA11 genes have emerged from a gene duplication, which has taken place either after the separation of the fish and tetrapod lineages or before this separation, with a subsequent loss of CA11 in the fish lineage (16).
13.7 CONCLUSIONS Although the CARPs are the least investigated a-CA isoforms, probably due to their lack of catalytic activity, the last years saw important advances in our understanding of these proteins. Most of the phylogeny related to these proteins is now understood in great detail through the excellent work from Parkkila and coworkers, who analyzed these relationships in all organisms for which the genomes are available so far (15–17). The expression pattern of the CARPs in human and murine tissues is also well understood, as well as the involvement of CARP VIII in genetic diseases, that is, ataxia, mental retardation, and quadrupedal gait in humans and lifelong gait disorder in humans and mice (20). Furthermore, in some tumors an overexpression of CAs VIII, X, and XI has been observed (20,35). However, the mechanism by which these proteins exert their important biological functions is still unknown. Interestingly, it has been also reported that restoring the putative zinc ligands in these three proteins leads to highly catalytically active enzymes for the CO2 hydration reaction. Overall, the CARPs deserve more detailed studies, mainly for understanding their biochemical and physiological functions in healthy and pathologic conditions.
REFERENCES 1. Grishin NV. Two tricks in one bundle: helix–turn–helix gains enzymatic activity. Nucleic Acids Res 2000;28:2229–33. 2. Todd AE, Orengo CA, Thornton JM. Evolution of protein function, from a structural perspective. Curr Opin Chem Biol 1999;3:548–56. 3. Andrade MA, Perez-Iratxeta C, Ponting CP. Protein repeats: structures, functions, and evolution. J Struct Biol 2001;134:117–31. 4. Jorda J, Kajava AV. Protein homorepeats sequences, structures, evolution, and functions. Adv Protein Chem Struct Biol 2010;79:59–88. 5. Kinch LN, Grishin NV. Evolution of protein structures and functions. Curr Opin Struct Biol 2002;12:400–8. 6. Alterio V, Di Fiore A, D’Ambrosio K, Supuran CT, De Simone G. Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? Chem Rev 2012;112:4421–68. 7. Supuran CT. Carbonic anhydrases as drug targets—an overview. Curr Top Med Chem 2007;7:825–33. 8. Supuran CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81. 9. Temperini C, Scozzafava A, Supuran CT. Carbonic anhydrase activation and the drug design. Curr Pharm Des 2008;14:708–15.
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10. Pastorekova S, Parkkila S, Pastorek J, Supuran CT. Carbonic anhydrases: current state of the art, therapeutic applications and future prospects. J Enzyme Inhib Med Chem 2004;19:199–229. 11. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for medicinal chemistry. Bioorg Med Chem 2007;15:4336–50. 12. Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors. Med Res Rev 2003;23:146–89. 13. Supuran CT,Vullo D, Manole G, Casini A, Scozzafava A. Designing of novel carbonic anhydrase inhibitors and activators. Curr Med Chem Cardiovasc Hematol Agents 2004;2:49–68. 14. Supuran CT,Vullo D, Manole G, Casini A, Scozzafava A. Designing of novel carbonic anhydrase inhibitors and activators. Curr Med Chem Cardiovasc Hematol Agents 2004;2:51–70. 15. Aspatwar A, Tolvanen ME, Ortutay C, Parkkila S. Carbonic anhydrase related proteins: molecular biology and evolution. Subcell Biochem 2014;75:135–56. 16. Aspatwar A,Tolvanen ME, Parkkila S. Phylogeny and expression of carbonic anhydrase-related proteins. BMC Mol Biol 2010;11:25. 17. Aspatwar A,Tolvanen ME, Parkkila S. An update on carbonic anhydrase-related proteins VIII, X and XI. J Enzyme Inhib Med Chem 2013;28:1129–42. 18. Nishimori I,Vullo D, Minakuchi T, Scozzafava A, Capasso C, Supuran CT. Restoring catalytic activity to the human carbonic anhydrase (CA) related proteins VIII, X and XI affords isoforms with high catalytic efficiency and susceptibility to anion inhibition. Bioorg Med Chem Lett 2013;23:256–60. 19. Aspatwar A, Tolvanen ME, Jokitalo E, Parikka M, Ortutay C, Harjula SK, et al. Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish. Hum Mol Genet 2013;22:417–32. 20. Aspatwar A,Tolvanen ME, Ortutay C, Parkkila S. Carbonic anhydrase related protein VIII and its role in neurodegeneration and cancer. Curr Pharm Des 2010;16:3264–76. 21. Elleby B, Sjoblom B, Tu C, Silverman DN, Lindskog S. Enhancement of catalytic efficiency by the combination of site-specific mutations in a carbonic anhydrase-related protein. Eur J Biochem 2000;267:5908–15. 22. Picaud SS, Muniz JR, Kramm A, Pilka ES, Kochan G, Oppermann U, et al. Crystal structure of human carbonic anhydrase-related protein VIII reveals the basis for catalytic silencing. Proteins 2009;76:507–11. 23. Hirota J, Ando H, Hamada K, Mikoshiba K. Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1. Biochem J 2003;372:435–41. 24. Kato K. Sequence of a novel carbonic anhydrase-related polypeptide and its exclusive presence in Purkinje cells. FEBS Lett 1990;271:137–40. 25. Taniuchi K, Nishimori I, Kohsaki T, Miyaji E, Okamoto N, Onishi S, et al. A discrepant finding between magnetic resonance imaging and other imaging modalities suggests a microcystic serous cystadenoma of the pancreas. Int J Pancreatol 2000;28:239–42. 26. Taniuchi K, Nishimori I, Takeuchi T, Fujikawa-Adachi K, Ohtsuki Y, Onishi S. Developmental expression of carbonic anhydrase-related proteins VIII, X, and XI in the human brain. Neuroscience 2002;112:93–9. 27. Bellingham J, Gregory-Evans K, Gregory-Evans CY. Sequence and tissue expression of a novel human carbonic anhydrase-related protein, CARP-2, mapping to chromosome 19q13.3. Biochem Biophys Res Commun 1998;253:364–7. 28. Nogradi A, Jonsson N, Walker R, Caddy K, Carter N, Kelly C. Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice. Brain Res Dev Brain Res 1997;98:91–101. 29. Nishimori I. Acatalytic CAs: carbonic anhydrase-related proteins. Boca Raton: CRC Press; 2004. 30. Morimoto K, Nishimori I, Takeuchi T, Kohsaki T, Okamoto N, Taguchi T, et al. Overexpression of carbonic anhydrase-related protein XI promotes proliferation and invasion of gastrointestinal stromal tumors.Virchows Arch 2005;447:66–73. 31. Bergenhem NC, Hallberg M, Wisen S. Molecular characterization of the human carbonic anhydraserelated protein (HCA-RP VIII). Biochim Biophys Acta 1998;1384:294–8. 32. Sjoblom B, Elleby B, Wallgren K, Jonsson BH, Lindskog S. Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme. FEBS Lett 1996;398:322–5. 33. Supuran CT. Carbonic anhydrases—an overview. Curr Pharm Des 2008;14:603–14. 34. Supuran CT. Carbonic anhydrase inhibitors. Bioorg Med Chem Lett 2010;20:3467–74. 35. Bataller L, Sabater L, Saiz A, Serra C, Claramonte B, Graus F. Carbonic anhydrase-related protein VIII: autoantigen in paraneoplastic cerebellar degeneration. Ann Neurol 2004;56:575–9.
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Acatalytic Carbonic Anhydrases (CAs VIII, X, XI) Claudiu T. Supuran*,**, Clemente Capasso*** *Università
degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Sesto Fiorentino, Florence, Italy Department, Sezione di Scienze Farmaceutiche, Sesto Fiorentino, Florence, Italy di Bioscienze e Biorisorse, CNR, Napoli, Italy
**NEUROFARBA ***Istituto
Contents 13.1 Introduction 239 13.2 Primary sequence analysis 240 13.3 Three-dimensional structure analysis 240 13.4 Physiological function and tissue distribution of CAs VIII, X, and XI 242 13.5 CARPs’ biochemical properties after restoring the catalytic site 242 13.6 Phylogenetic analysis 243 13.7 Conclusions 244 References 244
13.1 INTRODUCTION Most structural and regulatory proteins in eukaryotes are members of a gene family. Over the course of evolution, some duplicate genes were short-lived, losing functionality, and ultimately were removed (1–5). Enzymes having a chemical requirement for invariant amino acids in the active site are particularly vulnerable to selection pressure. The positive selection more often acts on residues adjacent to, rather than directly at, a critical active site of an enzyme, and on flexible regions rather than on rigid structural elements of the protein (1,2,5). This pattern might be a general mechanism for functional diversification of enzyme families, as it allows the acquisition of new functions without disrupting the native folding structure and primary enzyme function. But the natural evolution of enzymes can eventually lead to the formation of a new active site on the protein framework, or by transformation of an old active site for a new function. Interestingly, using sequence similarity programs, it is possible to identify proteins evolutionarily related to enzymes but lacking catalytic activity due to disruption of their active site. Most of these proteins develop a new function, for example, as transcription regulators (2,4,5). In this case, the direction of the evolution is simply that the enzyme lost its activity but acquired a new function.
Carbonic Anhydrases as Biocatalysts. DOI: 10.1016/B978-0-444-63258-6.00013-5 Copyright © 2015 Elsevier B.V. All rights reserved
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Carbonic anhydrases (CAs) are found in almost all organisms and are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3− (6–9). In mammals, a-CA gene family consists of 13 isoforms that are catalytically active and 3 isoforms that are catalytically inactive lacking the CO2-hydratase activity as well as other known catalytic functions normally associated with these enzymes, such as the esterase activity (7,10–14). Due to the high sequence and structural similarity with the active a-CAs, these proteins have been designated as CA-related proteins (CARPs) and were originally identified by screening Purkinje cell–specific genes (7,15–18).The three mammalian CARPs are named as CARP VIII, CARP X, and CARP XI. CARPs are predicted to be composed of 291 amino acids, and have an acidic amino acid cluster of 16 Glu and 4 Asp residues within the N-terminal 50 amino acid residues (18–20).These a-isoforms have a central CA motif, but the catalytic inactivity is due to lack of one or more of the three histidine residues that are necessary for coordination of the zinc ion within the active site (18,21).The enzymatic activity of mammalian CARP isoforms can be regained by restoration of these histidine residues (18). In the family of protein tyrosine phosphatases (PTPs), there are two receptor-type PTPs, -PTPR and g-PTPR, also containing an N-terminal CA-like domain, called “CARP XVI domain” (15,17). The physiological roles of CARPs are poorly understood for the moment. Here, we account the recent data on CARPs VIII, X, and XI.
13.2 PRIMARY SEQUENCE ANALYSIS Although the CARPs show a high degree of homology with the other known a-CA isoforms, they distinguish themselves from the catalytically active CAs by a salient feature: they lack one, two, or all three zinc protein ligands from the enzyme active site, that is, His94, His96, and His119 (the hCA I numbering system is used throughout this work) (15,17–19). Figure 13.1 shows the sequence alignment of hCA II with CARP VIII, CARP X, and CARP XI (denominated from now on as hCAs VIII, IX, and XI, respectively). Residue 94 is not a His, but an Arg in all the acatalytic isoforms; residue 96 is His in hCA VIII and hCA X, but Leu in hCA XI, whereas residue 119 is His only in hCA VIII, being a glutamine in hCA X and XI.Thus, in the acatalytic isoforms, one (hCA VIII), two (hCA X), and three (hCA XI) of the protein zinc ligands are changed from His (conserved in all the other catalytically active isoforms) to another amino acid, which normally has no possibility to coordinate the catalytically crucial zinc ion within the active site (18).
13.3 THREE-DIMENSIONAL STRUCTURE ANALYSIS In 2009, Oppermann and coworkers (22) solved the X-ray crystal structure of hCA VIII at high resolution, showing that the three-dimensional fold of the protein is rather similar to that of hCA II, the most studied and physiologically dominant
Acatalytic Carbonic Anhydrases (CAs VIII, X, XI)
Figure 13.1 Sequence alignment of hCA II (accession number: AAH11949.1), hCA VIII (accession number: AAI08930.1), hCA X (accession number: AAH47456.1) and hCA XI (accession number: AAH02662.1). The catalytic residues His94, His96 and His119 in hCA II and the corresponding residues of the acatalytic CARPs are underlined. The proton shuttle amino acid (His64) is conserved in all these molecules, while the gate keeper residues (Glu106 and Thr199) are conserved only in hCA II, hCA VIII and hCA X. Legend: (*) indicates identity at all aligned positions; (:) relates to conserved substitutions; (.) means that semi-conserved substitutions are observed. The hCA I numbering system was used. Multialignment was performed with the program MUSCLE, version 3.7
catalytically active isoform. As indicated in Figure 13.2A, a chloride ion has been found in the position where the Zn(II) ion is present in the catalytic CAs, such as hCA II (Figure 13.2B).This anion participates in two hydrogen bonds with His96 and His119, and another one with a water molecule, situated in a position, which is very similar to that of the zinc-coordinated water molecule in hCA II. Arg94 from hCA VIII had no interactions either with the chloride anion or with the above-mentioned water molecule, but the shape of the “active site” cavity was very similar to that of hCA II (18,22).
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Figure 13.2 (A) Ribbon diagram of hCA VIII structure. Relevant residues (His96, His119, and Arg94), Cl− ion (gray sphere), and the hydrogen-bonded water molecule are also shown. (B) Ribbon diagram of hCA II structure (PDB code 1CA2). The three histidines, the zinc ion (gray sphere), and the zinc bound water molecule are shown.
13.4 PHYSIOLOGICAL FUNCTION AND TISSUE DISTRIBUTION OF CAs VIII, X, AND XI The physiological role of some of these proteins, which are highly conserved in mammals, seems to be quite important (10,12–14,18). Thus, CA VIII has been initially identified in the brain, mainly in the Purkinje cells, but its expression has also been demonstrated in several other organs, such as the liver, lung, heart, gut, thymus, and kidneys (16,20,23–26). CAs X and XI are also predominantly found in the brain. The strong expression of CA VIII in the cerebellum suggested a role in brain function (10,19,20). This has been indeed documented later (27,28), when a mutation in the CA VIII gene was shown to be associated with ataxia, mild mental retardation, and quadrupedal gait in humans, and with lifelong gait disorder in mice, proving the important, yet poorly understood physiological role of this protein. Furthermore, CA VIII has been reported to be overexpressed in certain tumors, including non-small lung cancer and colorectal cancer (10,19,20). CA VIII mRNA was significantly expressed in developing human lungs but to a much lesser extent in normal adult lungs (29).These findings suggest that CA VIII plays a role in cell proliferation and carcinogenesis, in at least lung and colorectal epithelial cells. CA XI has also been observed to be overexpressed in gastrointestinal stromal tumors, promoting their proliferation (30). These data strongly suggest that CARPs may possess important physiological roles, poorly understood at this moment.
13.5 CARPs’ BIOCHEMICAL PROPERTIES AFTER RESTORING THE CATALYTIC SITE Recombinant proteins of human and mouse CA VIII have been reported to lack not only CO2 hydration activity but also esterase activity toward 4-nitrophenyl acetate as substrate (18). It is of interest that the single mutagenesis at position 94 from arginine to
Acatalytic Carbonic Anhydrases (CAs VIII, X, XI)
histidine was shown to restore the CO2 hydration activity. Further mutations, leading to a more “CA-like” active site cavity (e.g., Arg94His, Glu92Gln, Ile121Val, Ile143Val), resulted in a further increase of the catalytic activity of the mutant CA VIII (18). Sitedirected mutagenesis experiments in all three CARPs, as reported earlier for CA VIII by Lindskog and coworkers and Bergenhem et al. (21,31,32), were then performed, restoring their zinc-binding His residues. Interestingly, the recombinant hCAs VIII, X, and XI possessed a high catalytic activity for the physiological reaction, CO2 hydration to bicarbonate and protons, with the following kinetic parameters: kcat ranging from 7.20 × 105 to 1.15 × 106 s−1, KM in the range of 7.1–8.3 mM, and kcat/KM in the range of 1.01 × 108 to 1.38 × 108 M−1 s−1 (18). hCA X was found to be the most active among the investigated new enzymes. It was 92.0% as effective as hCA II as a catalyst for the physiological reaction. hCA XI showed a catalytic activity intermediate between that of hCA VIII (the least active) and hCA X (the most active isoform), being 82.6% as active as hCA II.The excellent catalytic activity of the mutated CAs VIII, X, and XI, much higher than that of hCA I, may be explained considering that in these enzymes (after the change of the residues in positions 94, 96, and 119 to His residues), the proton shuttle residue (His64) is also present. Restored CARPs were inhibited in the millimicromolar range by inorganic anions, sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid (18). Among the three new isoforms, hCA X showed the highest affinity for anion inhibitors (KI of 3.6–68 mM for phenylboronic acid, sulfamic acid, sulfamide, cyanide, and azide). hCA VIII was poorly inhibited by halides, cyanate, nitrate, and sulfate (KI of 38.4–65.4 mM), whereas CA XI had a behavior intermediate between that of hCA VIII and X, regarding the sensitivity to anion inhibitors (18). Probably the anions investigated in that study bind to these enzymes in a similar manner as they bind to other a-CAs investigated in more detail, coordinating to the metal ion, with the Zn(II) ion in either tetrahedral or trigonal bipyramidal geometries (33,34).
13.6 PHYLOGENETIC ANALYSIS CARP-like sequences have been identified in mammals and invertebrates. Vaccinia virus transmembrane protein D8 was reported to contain a CA-like N-terminal domain. CARP VIII sequences have been discovered in mammals and in many deuterostome invertebrates, such as mollusks and chordates, but not in protostomes, such as insects (16,20). CARP X–like proteins are present in vertebrate and invertebrate. In the invertebrate group, CARP X–like proteins are present even in nematodes and insects. CARP XI, the last to be identified, was recognized only in tetrapod vertebrates (15,17). Recently, Parkkila and coworkers reported that there is no ortholog of CA11 in the known fish genomes, but instead there are two descendants of CA10, namely, CA10A and CA10B12. This group carried out a phylogenetic analysis on all CARPs. They found that the origin of CA8 is more ancient than the origins of CA4, CA5, CA6, CA9, CA12, and CA14, which were formed during the radiation of jawed vertebrates, or CA1, CA2, CA3, and CA13, which are specific to the tetrapod lineage (15,17,19).
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Carbonic Anhydrases as Biocatalysts
Phylogenetic analysis of CARP X sequences showed that they form two groups: one consisted of sequences from mammals, frogs, and lizards; and the other, formed by two smaller groups, consisted of sequences from fishes (17). CARP XI phylogenetic analysis suggests that the CA11 genes have emerged from a gene duplication, which has taken place either after the separation of the fish and tetrapod lineages or before this separation, with a subsequent loss of CA11 in the fish lineage (16).
13.7 CONCLUSIONS Although the CARPs are the least investigated a-CA isoforms, probably due to their lack of catalytic activity, the last years saw important advances in our understanding of these proteins. Most of the phylogeny related to these proteins is now understood in great detail through the excellent work from Parkkila and coworkers, who analyzed these relationships in all organisms for which the genomes are available so far (15–17). The expression pattern of the CARPs in human and murine tissues is also well understood, as well as the involvement of CARP VIII in genetic diseases, that is, ataxia, mental retardation, and quadrupedal gait in humans and lifelong gait disorder in humans and mice (20). Furthermore, in some tumors an overexpression of CAs VIII, X, and XI has been observed (20,35). However, the mechanism by which these proteins exert their important biological functions is still unknown. Interestingly, it has been also reported that restoring the putative zinc ligands in these three proteins leads to highly catalytically active enzymes for the CO2 hydration reaction. Overall, the CARPs deserve more detailed studies, mainly for understanding their biochemical and physiological functions in healthy and pathologic conditions.
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